Poly(amino acids), including peptides and proteins, are typically detected and characterized using gel electrophoresis, by solution quantitation assays or by detection on solid supports, such as filter membranes. Small amounts of protein or other poly(amino acids) are generally not visible to the naked eye, and must be stained before they can be localized and identified.
Two of the most common methods of staining poly(amino acids) in gels are COOMASSIE Brilliant Blue (CBB) staining and silver staining. For particular poly(amino acids), silver staining is approximately 100- to 1000-fold more sensitive than CBB staining, but both methods have disadvantages. The use of luminescent reagents for protein detection offers the possibility of greatly enhanced sensitivity and increased linear quantitation range, while simultaneously increasing the ease of use of the staining reagent. By "luminescent" is meant any reagent that is fluorescent, phosphorescent or chemiluminescent.
The use of selected styryl and merocyanine dyes as fluorescent stains for poly(amino acids) in gels (e.g. SYPRO.RTM. Red and SYPRO.RTM. Orange dyes of Molecular Probes, Inc.), on membranes or other supports, and in solution (U.S. Pat. No. 5,616,502 to Haugland et al., hereby incorporated by reference) is very rapid, relatively insensitive to poly(amino acid) composition, does not require destaining, and is more than an order of magnitude more sensitive than CBB staining.
Unfortunately, organic fluorescent dyes typically suffer from the drawback of high background noise. Europium complexes of sulfonated bathophenanthroline that permit "time-resolved" detection reduce the interference from background noise (see M. J. Lim, et al., ANAL. BIOCH. 245, 184-195 (1997), and International Publication No. WO 97/20213). However, the europium complexes are not readily excited by visible light, and require ultraviolet illumination for optimal luminescence, thus they cannot be used with commercially available laser-excited gel scanners. In addition, the europium complexes bind to proteins reversibly, and decompose at low concentrations.
Some dyes have been used to stain proteins using so-called "colloidal" dye formulations. A restricted number of sulfonated triphenylmethane dyes are known to form colloidal protein-dye complexes. Other sulfonated dyes do not selectively stain proteins in polyacrylamide gels when prepared in such formulations.
The transition metal complexes used to practice the method of the instant invention have an overall charge that is neutral, or that is anionic. The complexes are highly stable, even in dilute solution, associate strongly with proteins in solution, on membranes, in biological cells, and in electrophoretic gels, yielding bright, long-lifetime, visible luminescence. The instant metal complexes bind strongly and noncovalently to proteins, even in neutral or basic pH solutions, and provide higher sensitivity poly(amino acid) detection than any of the methods described above, and can be used with both ultraviolet and with visible light excitation.